Acyl-homoserine lactone (HSL) quorum sensing occurs in a wide variety of Gram-negative bacterial species. Details of the signals, signal generators and signal receptors have been generated at a rapid pace in the past several years. Quorum sensing functions as a global regulator of gene expression and the investigators are just beginning to explore this aspect of the signaling. Pseudomonas aeruginosa is a particularly useful model to study global quorum control of gene expression. The genome has been sequenced, there is a commercial GeneChip, and libraries of mapped insertion mutants are being created. The signals and signal genes have been identified, and the applicants have identified over 300 genes in the quorum-controlled "regulon." P. aeruginosa quorum sensing is particularly relevant as it is required for virulence of this emerging human pathogen. The goals of this project are as follows: I. To fine map the induction of "early" quorum-controlled genes. This analysis will be a step in testing the hypothesis that the strength of the binding sites for the transcriptional activators defines the precise timing of induction. II. To engineer P. aeruginosa strains with investigator-controllable LasR and RhlR expression modules and use the constructs to test the hypothesis that at high levels of expression the timing of all genes directly regulated by LasR or RhlR should be early. At low levels of expression only those promoters with the greatest affinity for the transcription factors should respond to signal addition. III. To study the signal receptors (transcription factors) in vitro to test models for mechanisms, which control the complex quorum gene regulatory patterns. IV. To study one particular quorum-controlled gene that is specifically expressed in stationary phase. V. To examine the role of quorum sensing in biofilm development. The P. aeruginosa quorum sensing system provides an opportunity to understand how a simple regulatory element can affect an ordered expression of hundreds of genes in a bacterial culture. The proposed research will serve to enrich the understanding of the mechanisms and physiological significance of quorum sensing. [unreadable] [unreadable]